Expression vectors containing λPL promoter, T1 T2 rRNA termination sequence and origin of replication derived from a constitutive high copy number plasmid hosts and related methods

ABSTRACT

An improved vector upon introduction into a suitable host containing the thermolabile repressor C I  renders the host capable of effecting expression of a desired gene. The vector is a double-stranded DNA molecule which includes in 5&#39; to 3&#39; order the following: the promoter and operator P L  O L  from lambda bacteriophage; the N utilization site; a first restriction enzyme site permitting replacement of the ribosomal binding site which follows thereafter; a ribosomal binding site for transcribing mRNA; an ATG initiation codon or DNA which is converted into an ATG initiation codon upon insertion of the desired gene into the vector; a second restriction enzyme site for inserting the gene in phase with the ATG codon; a T 1  T 2  rRNA transcription termination sequence; a DNA sequence which contains an origin of replication capable of autonomous production in the host of at least 400 constitutive copies of the vector; and either a gene associated with a selectable or identifiable phenotype trait manifested when the vector is present in the host cell or the fragment cI 434  on which is included the gene for the repressor protein and its associated promoter and operator. The distance between the 3&#39; end of P L  O L  and the 5&#39; end of the N utilization site is less than about 80 base pairs. The distance between the 3&#39; end of the N utilization site and the 5&#39; end of the ribosomal binding site is less than about 300 base pairs. 
     Plasmids have been constructed from the vectors and used to produce bovine growth hormones.

This is a continuation of application Ser. No. 645,119, filed Aug. 27,1984, now abandoned.

BACKGROUND OF THE INVENTION

One aspect of genetic engineering involves the insertion of foreign DNAsequences derived from eucaryotic sources into Escherichia coli or othermicroorganisms. A further refinement of genetic engineering concernsinducing the resulting microorganism to produce polypeptides encoded bythe foreign DNA. Production of polypeptides can be considered a two-stepprocess, with each step including numerous substeps. The two steps aretranscription and translation. To produce a polypeptide efficiently andin quantity both steps of the process must be efficient. Transcriptionis the production of mRNA from the gene (DNA). Translation is theproduction of polypeptide from the mRNA.

A critical substep of the transcription process is initiation, that is,the binding of RNA polymerase to a promoter-operator region. Thesequence of deoxyribonucleotide bases which make up the promoter regionmay vary and thereby effect the relative efficiency of the promoter. Theefficiency depends on the affinity of the RNA polymerase for thepromoter.

The efficiency of translation is affected by the stability of the mRNA.Increased stability of the mRNA permits improved translation. Althoughthe exact determinants of mRNA stability are not precisely known, it isknown that mRNA secondary structure as determined by the sequence of itsbases has a role in stability.

The initial substep of translation involves binding of the ribosome to abase sequence on the mRNA known as the Shine-Dalgarno sequence or theribosomal binding site (RBS). The synthesis of polypeptides begins whenthe ribosome migrates along the mRNA to the AUG start codon fortranslation. Generally these codons are found approximately 10 bases"downstream" from the Shine-Dalgarno site. Factors which increase theefficiency of translation include those which enhance binding of theribosomes to the Shine-Dalgarno site. It has been shown that thestructure of the mRNA in the region of the Shine-Dalgarno sequence andthe AUG codon and the distance between the Shine-Dalgarno sequence andthe AUG codon each play a critical role in determining the efficiency oftranslation. Other factors which affect the efficiency of translationare premature termination and attenuation. Efficiency of translation canbe improved by removing the attenuation sites.

A difficulty encountered in attempts to produce high amounts ofeucaryotic polypeptides in bacterial cells involves the inability ofcells producing large amounts of mRNA to grow efficiently. Thisdifficulty can be eliminated by preventing transcription by a processknown as repression. In repression genes are switched off due to theaction of a protein inhibitor (repressor protein) which preventstranscription by binding to the operator region. After microorganismshave grown to desired cell densities, the repressed genes are activatedby destruction of the repressor or by addition of molecules known asinducers which overcome the effect of the repressor.

Numerous reports may be found in the literature concerning the cloningof eucaryotic genes in plasmids containing the P_(L) promoter from λbacteriophage. (Bernard, H. V., et al., Gene (1979) 5, 59; Derom, C., etal., Gene (1982) 17, 45; Gheysen, D., et al., Gene (1982) 17, 55;Hedgpeth, J. et al., Mol. Gen. Genet. (1978) 163, 197; Remaut, E., etal., (1981) Gene 15, 81 and Derynck, R., et al., Nature (1980) 287, 193.In addition, European Patent Application No. 041,767, published Dec. 16,1981, describes expression vectors containing the P_(L) promoter from λbacteriophage. However, none of these references describe the use of theC_(II) ribosomal binding site.

The use of a vector containing the P_(L) promoter from λ bacteriophageand the C_(II) ribosomal binding site has been described. (Oppenheim, A.B. et al., J. Mol. Biol. (1982) 158, 327 and Shimatake, H. andRosenberg, M., Nature (1981) 292, 128.) These publications describe theproduction of increased levels of C_(II) protein but do not involve ordescribe the production of eucaryotic proteins.

Other vectors which contain the P_(L) promoter and the C_(II) ribosomalbinding site have also been described (Courntey, M. et al., PNAS (1984)81, 669-673; Lautenberger, J. A. et al., Gene (1983) 23, 75-84 andLautenberger, J. A. et al., Science (1983) 221, 858-860). However, allof these vectors lead to the production of fused proteins which containthe amino terminal portion of the C_(II) protein.

In 1982 Shatzman and Rosenberg presented a poster at the 14th MiamiWinter Symposium (Shatzman, A. R. and Rosenberg, M., 14 Miami WinterSymposium, abstract p98 [1982]). This abstract provides a non-enablingdisclosure of the use of a vector containing P_(L) from λ bacteriophage,Nut and the C_(II) ribosomal binding site to synthesize a "eucaryotic"polypeptioe (SV40 small T antigen is actually not a eucaryoticpolypeptide but a viral protein) in an amount greater than 5% of thecell protein in an unnamed bacterial host. The operator used is notdefined. Neither an origin of replication nor a gene for a selectablephenotype is identified. This system with which the vector is used isdescribed as including certain host lysogens into which the vector canbe stably transformed.

Applicants are aware of the existence of a pending U.S. patentapplication in the name of M. Rosenberg filed under Ser. No. 457,352 bythe National Institutes of Health, Dept. of Health and Human Services,U.S.A. Portions of this application have been obtained from the NationalTechnical Information Service, U.S. Dept. of Commerce. However, theclaims are not available and are maintained in confidence. The availableportions of the application have been reviewed. This disclosure is notenabling. It indicates that the host is important (p8, line 17) butfails to identify any suitable host. It further depends upon the use ofa λ mutant which is not specified (p4, line 20). It indicates that thehost contains lysogens (p8, line 18) unlike the present invention inwhich the host is not lysogenic. It mentions cloning and expression of aeucaryotic gene, monkey metallothionein gene, (p7, line 18) but does notprovide details. It specifies that neither the sequence nor the positionof any nucleotide in the C_(II) ribosomal binding region has beenaltered (p3, line 27).

Pending, co-assigned U.S. patent application Ser. No. 514,188, filedJuly 15, 1983, describes novel vectors useful for the expression ofpolypeptides in bacteria. These vectors include P_(L) O_(L), Nutilization site for binding antiterminator N protein, ribosomal bindingsite, ATG codon, restriction enzyme site for inserting the gene encodingthe desired polypeptide, an origin of replication and a selectablemarker. In these vectors the distance between the N utilization site andthe ribosomal binding site is greater than about 300 base pairs. Inaddition, each of these vectors contains a specific ribosomal bindingsite which cannot be readily replaced. These vectors were not equallyuseful for expression of different polypeptides.

T₁ T₂ rRNA transcription termination sequences have been described(Brosius, J., et al., J. Mol. Biol. 148, 107 (1981)). The placement ofT₁ T₂ rRNA termination sequences at the 3' end of a procaryotic gene andthe expression of such gene under the control of a promoter have beendescribed (Amann, E., et al., Gene (1983) 25, 167; Zabeau, M., et al.,The EMBO Journal (1982) 1, 1217).

European patent application no. 81304573.9, published April 14, 1982under European publication no. 049,619, discloses the use of the λcI857thermoinducible repressor as a stabilizing element. The repressor iscloned on the plasmid. A λcI90 prophage defective in repressor synthesisis introduced by infection. The prophage is maintained by the clonedrepressor at temperatures below 32° C. Any cell losing the plasmid willbe lysed. If the temperature is increased to above 38° C., the repressoris destroyed or inactivated and the cells lyse. This stabilizationsystem is not compatible with the vectors of the invention which includeλP_(L) promoter and which express polypeptides at temperatures above 38°C.

Origins of replication from constitutive high copy number plasmids areknown. For example pOP1Δ6 origin of replication from ColEl has beendescribed (Gelfand, D. H., et al., PNAS (1978) 75, (12), 5869 andMuesing, M., et al. Cell (1981) 45, 235). In addition, high copy numberrun-away replication plasmids, as distinguished from, constitutive highcopy number plasmids, are known (Remant, E., et al. Gene (1983) 22,103).

The present invention relates to expression vectors which unexpectedlyprovide enhanced expression of different polypeptides. By employingdifferent ribosomal binding sites in the vectors of this invention it ispossible to achieve enhanced expression levels of different polypeptidesrelative to the levels achieved with the previous vectors. In addition,using the same ribosomal binding sites as in the previous vectors, it ispossible to achieve enhanced expression of the same polypeptides.Moreover, by placing T₁ T₂ rRNA termination sequences at the 3' end ofthe gene encoding a polypeptide whose expression is desired, it ispossible to increase the amount of desired polypeptide relative to thetotal polypeptide produced by a bacterial host. As importantly, thepresence of the T₁ T₂ rRNA transcription termination sequences permitorigins of replication derived from constitutive high copy numberplasmids to be incorporated into expression vector without loss of theability to replicate in constitutive high copy number.

Origin of replication derived from pBR322 or nonconstitutive high copynumber plasmids other than runaway high copy number plasmids whenincorporated into a vector are capable of producing only a certainnumber of copies per cell, typically less than 40 copies per cell. Bysubstituting an origin of replication from a constitutive high copynumber plasmid it has unexpectedly been found that that level ofpolypeptide expression is increased.

The preferred vectors of this invention are stabilized in the bacterialhost and when bacteria containing plasmids which include the vectors andgenes encoding polypeptides are grown, the plasmids are not lost. Inthis way, yield reduction caused by plasmid instability is overcome.Moreover, use of such preferred vectors avoids the use of antibioticresistance as a selectable marker, thus permitting lower costs forproducing polypeptides.

SUMMARY OF THE INVENTION

This invention concerns an improved expression vector which uponintroduction into a suitable bacterial host cell, namely, Escherichiacoli, containing the thermolabile repressor C_(I) renders the host cellcapable, upon increasing the temperature of the host cell to atemperature at which the repressor is destroyed, of effecting expressionof a desired gene inserted into the vector and production of thepolypeptide encoded by the gene comprising:

a double-stranded DNA molecule which includes in 5' to 3' order thefollowing:

a DNA sequence which contains the promoter and operator P_(L) O_(L) fromlambda bacteriophage;

the N utilization site for binding antiterminator N protein;

a first restriction enzyme site permitting replacement of the DNAsequence containing the ribosomal binding site which follows thereafter;

a DNA sequence which contains a ribosomal binding site for rendering themRNA of the desired gene capable of binding to ribosomes within the hostcell;

an ATG initiation codon or a DNA sequence which is converted into an ATGinitiation codon upon insertion of the desired gene into the vector;

a second restriction enzyme site for inserting the desired gene into thevector in phase with the ATG initiation codon; and

a DNA Sequence which contains a T₁ T₂ rRNA sequence;

and which additionally includes a DNA sequence which contains an originof replication from a bacterial plasmid capable of autonomousreplication in the host cell and production of at least 400 constitutivecopies of the vector and either a DNA sequence which contains a geneassociated with a selectable or identifiable phenotypic trait which ismanifested when the vector is present in the host cell or a DNA sequencewhich contains the fragment designated cI⁴³⁴, such fragment includingthe gene for the cI⁴³⁴ repressor protein and its associated promoter andoperator sequence, the distance between the 3' end of the P_(L) O_(L)promoter and operator sequence and the 5' end of the N utilization sitebeing less than about 80 base pairs and the distance between the 3' endof the N utilization site and the 5' end of the ribosomal binding sitebeing less than about 300 base pairs. Desirably, the T₁ T₂ rRNAtermination sequence is less than about 100 base pairs from the 3' endof the second restriction enzyme site, more desirably it is less thanabout 20 base pairs from the 3' end of the site. Desirably the vectorincludes both the gene associated with the phenotypic trait and thecI⁴³⁴ fragment. More desirably, the cI⁴³⁴ fragment is located after the3' end of the T₁ T₂ rRNA termination sequence. The presently preferredorigin of replication is pOPIΔ6 which is derived from a ColEl plasmid.

Genes, e.g., cDNAs, encoding desired polypeptides, such as growthhormones, e.g., bovine, porcine, chicken or human growth hormones, humansuperoxide dismutase, human apolipoprotein E or analogs thereof, may beinserted into the second restriction enzyme site of the vector to createplasmids. The plasmids in turn can be introduced into suitable hostswhere the genes can be expressed and the desired polypeptide produced.The presently preferred plasmids for bovine growth hormone (bGH) arep8300-10A, pSAL-170/10 and pSAL-210/4. Preferred hosts includeEscherichia coli A1637, A1645, A2602, A2097 and A1563 if the plasmiddoes not contain the cI⁴³⁴ fragment and A1645 (λi434cI⁻ miniTn10) if theplasmid contains the cI⁴³⁴ fragment.

The resulting host vector systems can be employed to manufacturepolypeptides, e.g., growth hormones. Host cells containing the plasmidsare grown under suitable conditions permitting production of polypeptideand the resulting polypeptide is recovered.

DESCRIPTION OF THE FIGURES

The restriction maps for each of the plasmids shown in FIGS. 1-24 do notidentify all restriction sites present on each plasmid. In some casesrestriction sites are shown in one figure but not in another. However,in all cases those restriction sites necessary for a completeunderstanding of the invention are shown.

FIG. 1 Construction of pAL500

A plasmid containing bGH cDNA, D4 (ATCC No. 31826), was digested withHaeII. The resulting 1600 base pair large fragment was purified anddigested at 37° C. for 5 minutes with S1 exonuclease. A synthetic EcoRIlinker with the sequence: ##STR1## was attached to the ends of theresulting fragments by ligation. The ligation mixture was cleaved withEcoRI and inserted into pBR322 (ATCC No. 37017) which had been cleavedwith EcoRI. A clone, pALRI, was obtained which upon cleavage with EcoRIreleased a 1200 base pair fragment with the sequence: ##STR2## at the 5'end. This sequence demonstrates that pALRI contains an EcoRI restrictionsite which includes the TTC codon for residue number 1 (phenylalanine)of natural bGH. pALRI was subjected to a partial cleavage with PstI. Thedigest was treated with DNA polymerase I large fragment (Klenow) andHindIII linkers with the sequence: ##STR3## were attached by ligation.The ligation mixture was cleaved with EcoRI and HindIII. The fragmentcontaining bGH cDNA was isolated and subcloned into pBR322 between theEcoRI and HindIII restriction sites to give pAL500 (ATCC No.39782).

FIG. 2 Construction of pRO211 and pRO12

The plasmid pJH200 (ATCC No. 39783) was partially digested with NdeI,treated with DNA polymerase I (Klenow) to fill in the ends and theresulting ends were religated to form the expression vector pRO211. Theexpression vector pRO211 was digested with NdeI and HindIII, the largefragment isolated and ligated to an NdeI-HindIII bGH fragment isolatedfrom pAL500 (ATCC No. 39782) to give pRO12. (The NdeI-HindIII fragmentwas produced from pAL500 by digesting it with EcoRI and ligating to theends of the digestion product synthetic linkers with the sequence:##STR4## The ligation mixture was digested with NdeI and HindIII and theresulting NdeI-HindIII bGH fragment isolated.)

FIG. 3 Construction of pSAL 5200-6

pRO12 (FIG. 2) was partially digested with PvuII followed by digestionwith NdeI to eliminate a 72 base pair fragment. A synthetic DNA fragmentcoding for the first 24 amino acids of the N-terminus of authentic bGHwas ligated to the digested pRO12.

The synthetic DNA fragment was constructed by annealing twophosphorylated synthetic single-stranded DNAs of the sequence: ##STR5##The annealed fragment was treated with DNA polymerase I (Klenow) in thepresence of all four deoxyribonucleoside triphosphates in order to formthe full length double-stranded DNA. The fragment was digested withPvuII and NdeI before ligation to pRO12 to form pSAL 5200-6.

FIG. 4 Construction of p3008

p3008 (ATCC No. 39804) was constructed by ligating NdeI-digested pRO211[FIG. 2) with the pGH fragment isolated from an NdeI digest of theplasmid ppGH-NdeI/RI.

ppGH-NdeI/RI contains full length pGH cDNA to both ends of which NdeIsites have been added by means of synthetic linkers.

FIG. 5 Construction of p5002

p5002 was constructed by tripartite ligation of a dimerized syntheticlinker and the 2 cGH fragments isolated from an NdeI and BanII digest ofthe plasmid pcGH-NdeI/RI. The ligation mixture was digested with NdeIand then ligated to the expression vector pRO211 (FIG. 2) after it hadbeen restricted with NdeI. A colony containing the plasmid p5002 wasisolated.

The synthetic linker was constructed from two single-stranded syntheticDNAs of the sequence ##STR6##

The linker was phosphorylated before ligation. The linker codes for thefirst 18 amino acids of the N-terminus of the authentic cGH.

The plasmid pcGH-NdeI/RI contains full length cGH cDNA at the 5' end ofwhich there is an EcoRI restriction site and at the 3' end of whichthere is an NdeI restriction site. These restriction sites were added bymeans of synthetic linkers.

FIG. 5 Construction of pHG44 and pHG50

pRO12 (FIG. 2) was digested with HindIII. The linear form DNA (form III)was purified from agarose gel and ligated to a HindIII-HindIII fragmentof about 1200 base pairs which contains the rRNA operon transcriptiontermination sequences T₁ T₂. The T₁ T₂ HindIII-HindIII fragment wasisolated from plasmid pPS1 (ATCC No. 39807) which had been digested withHindIII. The resulting plasmid pHG44 (ATCC No. 39806) contains the T₁ T₂sequences at the 3' end of the recombinant (rec) bGH sequence.

The plasmid pSK434 (ATCC No. 39784) containing the λcI⁴³⁴ repressorsequences was digested with HpaII. The λcI⁴³⁴ HpaII-HpaII fragment wasisolated and ligated to pHG44 which had been digested with ClaI. Theresulting plasmid pHG50 (ATCC No. 39805) contains the T₁ T₂transcription termination sequences and the λcI⁴³⁴ repressor sequence.

FIG. 7 Construction of p8300-10A

The plasmid p8300-10A (ATCC No. 39785) which expresses an analog of thenatural phenylalanine form of bGH having methionine at the N-terminus(met--phe bGH) was prepared as follows. The plasmid p7200-22 containsthe λP_(L) promoter and ribosomal binding site derived from pJH200 (ATCCNo. 39783), DNA encoding met--phe bGH and the T₁ T₂ rRNA terminationsequences. ClaI--ClaI fragment containing the λP_(L) promoter, theC_(II) ribosomal binding site, the met--phe bGH gene and the T₁ T₂transcription termination sequences was inserted into the unique ClaIsite of plasmid pOP1Δ6, a constitutive high copy number plasmid, to formp830-10A.

FIG. 8 Construction of pSAL-130/5 and pSAL-170/10.

The plasmid pHG44 (ATCC No. 39806) expressing met--asp--gln bGH proteinwas digested with NdeI and HindIII. The resulting NdeI-HindIII bGHfragment was isolated and ligated to a fragment from p830-10A (ATCC No.39785) prepared by partial digestion with both NdeI and HindIII. Such aligation replaces the met--phe bGH gene fragment with the met--asp--glnbGH gene fragment. The plasmid so obtained, pSAL-130/5, expresses recbGH. pSAL-170/10 was obtained by treating the EcoRI--AvaI fragmentcontaining the Tet® gene of pBR322 plasmid (ATCC No. 37017) with DNApolymerase I (Klenow) and inserting it into pSAL-130/5 which had beendigested with BamHI and filled in with DNA polymerase I (Klenow).

FIG. 9 Construction of pSAL-210/4

Linear form DNA (form III) was prepared by partial ClaI digestion ofpSAL-170/10. It was purified from an agarose gel and ligated to aHpaII-HpaII cI⁴³⁴ gene fragment which was isolated from a HpaII digestof the plasmid pSK434 (ATCC No. 39784).

FIG. 10 Construction of pSAL 5600-1

pSAL 5200-6 (FIG. 3) was digested with HindIII. The linear form DNA(form III) was purified from an agarose gel and ligated to aHindIII-HindIII fragment of about 1200 base pairs which contains therRNA operon transcription termination sequences, T₁ T₂ The T₁ T₂HindIII-HindIII fragment was isolated from the plasmid pPS1 (ATCC No.39807) which was digested with HindIII. The resulting plasmid pSAL5600-1 contains the T₁ T₂ sequences at the 3' end of the met--asp--glnbGH sequence.

FIG. 11 Construction of p3009

The NdeI-NdeI pGH fragment was isolated from plasmid p3008 (ATCC No.39804) (FIG. 5). The fragment was inserted into the unique NdeI site ofthe expression vector p579 (FIG. 19) which had been digested with NdeI.The resulting plasmid p3009 expresses an analog of natural porcinegrowth hormone protein having a methionine residue added at theN-terminus.

FIG. 12 Construction of p5003

The NdeI-NdeI cGH fragment was isolated from plasmid p5002. The fragmentwas inserted into the unique NdeI site of the expression vector p579(FIG. 19) which had been digested with NdeI. The resulting plasmid p5003(ATCC No. 39792) expresses an analog of natural chicken growth hormoneprotein having a methionine residue added at the N-terminus.

FIG. 13 Construction of pSODc2

The pJH200 (ATCC No. 39783) expression vector was digested with NdeI.The 550 base pair NdeI fragment containing the λP_(L) promoter andC_(II) ribosomal binding site was isolated and inserted into the uniqueNdeI site of plasmid pSOD NH-10 which had been digested with NdeI.(Plasmid pSOD NH-10 is derived from a cDNA clone of human SOD[Lieman-Hurwitz, J., et al., PNAS (1982) 79:2808 ]) The resultingplasmid pSOD NH-550 was digested with AluI. (Only the relevant AluI siteis shown in the figure.) The large AluI fragment containing the λP_(L)promoter and the SOD gene was isolated. BamHI linkers were attached andthe resulting fragment was digested with BamHI. The BamHI digestionproduct was inserted into the unique BamHI site of pBRM (ATCC No. 37283)to form pSODα2 (ATCC No. 39786).

FIG. 14 Construction of pSODo13 and pSODβ1

The plasmid pSODα2 (ATCC No. 39786) was partially digested with EcoRIand the resulting linear form DNA was isolated from an agarose gel. Thepurified DNA was filled in with DNA polymerase I (Klenow) and religated.The resulting clone pSODα13 contains one EcoRI site located at the 5'end of the ribosomal binding site. A fragment containing the β-lactamasepromoter and ribosomal binding site was isolated from plasmid pBLAll(ATCC No. 39788) which had been digested with EcoRI and AluI. The 200base pair fragment was ligated to the large fragment isolated frompSODα13 which had been digested with NdeI, filled in with DNA polymeraseI (Klenow) and then digested with EcoRI. The resulting plasmid pSOD⊕1contains the ribosomal binding site of the β-lactamase gene and theλP_(L) promoter.

FIG. 15 Construction of pSODβ₁ T₁₁

Plasmid pBR322 (ATCC No. 37017) was digested with EcoRI and AvaI. Theresulting DNA was filled in with DNA polymerase I (Klenow). The Tet^(R)gene fragment was then isolated and ligated to the large fragmentisolated from pSODβ1 (FIG. 14) plasmid which had been digested with PstIfollowed by a partial BamHI digest and then filled in with DNApolymerase I (Klenow). The resulting plasmid pSODβ₁ T₁₁ contains theTetR gene.

FIG. 16 Construction of pSODβ₁ TT-1

The rRNA T₁ T₂ transcription termination fragment was isolated fromplasmid pPS1 (ATCC No. 39807) which had been digested with HindIII andfilled in with DNA polymerase I (Klenow;. The fragment was ligated toplasmid pSODβ₁ T₁₁ (FIG. 15) which had been partially digested withBamHI and filled in with DNA polymerase I (Klenow).

FIG. 17 Construction of pSODβ₁ --BA2

A synthetic DNA fragment with the sequence: ##STR7## which is similar tothe sequence of the natural β-lactamase ribosomal binding site, wasphosphorylated and ligated to the large fragment of pSODα13 plasmid(FIG. 14) which had been digested with NdeI and EcoRI.

FIG. 18 Construction of pTV-188

Plasmid pApoE-EX2 (ATCC No. 39787) was digested with NdeI and thenfragments filled in with DNA polymerase I (Klenow). The resulting ApoEgene fragment was isolated and inserted into the unique blunt end StuIsite of the pSODβ₁ T₁₁ plasmid (FIG. 15). The resulting plasmid pTV-188expresses an ApoE fused protein.

FIG. 19 Construction of p579

The rRNA operon T₁ T₂ transcription termination fragment was isolatedfrom plasmid pPS1 (ATCC No. 39807) which had been digested with HindIII.The T₁ T₂ fragment was inserted into the unique HindIII site of pRO211(FIG. 2) which had been digested with HindIII. The resulting expressionvector, p579, contains the λP_(L) promoter, the C_(II) ribosomal bindingsite, followed by the T₁ T₂ transcription termination signals.

FIG. 20 Construction of pTV-170

The NdeI-NdeI ApoE fragment was isolated from plasmid pApoE-EX2 (ATCCNo. 39787) and inserted into the unique NdeI site of the expressionvector p579 (FIG. 19) which had been digested with NdeI. The resultingplasmid pTV-170 expresses an analog of natural human ApoE protein havinga methionine residue added at the N-terminus.

FIG. 21 Construction of pTV-190

The plasmid pTV-170 (FIG. 20) was partially digested with NdeI andfilled in with DNA polymerase I (Klenow). The isolated linear form DNAwas religated to yield the plasmid pTV-190 which was analyzed and foundto have only one NdeI site at the 5' end of the ApoE gene.

FIG. 22 Construction of pTV-194

The β-lactamase promoter and ribosomal binding site fragment wasisolated from plasmid pBLAll (ATCC No. 39788) after digestion with EcoRIand AluI. This fragment was ligated to the large fragment of pTV-170(FIG. 20) plasmid which had been digested with NdeI, filled in with DNApolymerase I (Klenow) and then digested with EcoRI.

FIG. 23 Construction of pSAL 160-5

An AvaI-AvaI fragment containing the ApoE DNA sequence was isolated frompTV-170 (FIG. 21) which was digested with AvaI. The fragment was filledin with DNA polymerase I (Klenow) and isolated on agarose gel. Thepurified ApoE fragment was inserted into the PstI site cf the pTV 104(2)plasmid (ATCC No. 39384) which was partially digested with PstI andfilled in with DNA Polymerase I (Klenow). The resulting plasmid isdesignated pSAL 160-5.

FIG. 24 Construction of pTV-214

A synthetic fragment containing the first 14 amino acids of human growthhormone with the sequence: ##STR8## was phosphorylated using Y-³² P--ATPand polynucleotide kinase. The phosphorylated linker was inserted intothe unique NdeI site of pTV-190 plasmid which had been digested withNdeI.

DETAILED DESCRIPTION OF THE INVENTION

A vector has been developed which enables the achievement of enhancedlevels of gene expression and polypeptide production. The vector is adouble-stranded DNA molecule. Upon introduction into a suitablebacterial host cell containing the thermolabile repressor C_(I) thevector renders the host cell capable, upon increasing the temperature ofthe host cell to a temperature at which the repressor is inactivated, ofeffecting expression of a desired gene inserted into the vector andproduction of polypeptide encoded by the gene.

The vector includes in 5' to 3' order the following:

a DNA sequence which contains the promoter and operator P_(L) O_(L) fromlambda bacteriophage;

the N utilization site for binding antiterminator N protein;

a first restriction enzyme site permitting replacement of the DNAsequence containing the ribosomal binding site which follows thereafter;

a DNA sequence which contains a ribosomal binding site for rendering themRNA of the desired gene capable of binding to ribosomes within the hostcell;

an ATG initiation codon or a DNA sequence which is converted into an ATGinitiation codon upon insertion of the desired gene into the vector;

a second restriction enzyme site for inserting the desired gene into thevector in phase with the ATG initiation codon; and

a DNA sequence which contains a T₁ T₂ rRNA transcription terminationsequence.

The vector also includes a DNA sequence which contains an origin ofreplication from a bacterial plasmid capable of autonomous replicationin the host cell of at least 400 constitutive copies of the vector. Inaddition the vector includes either a DNA sequence which contains a geneassociated with a selectable or identifiable phenotypic trait which ismanifested when the vector is present in the host cell or a DNA sequencewhich contains the fragment designated cI⁴³⁴ The cI⁴³⁴ fragment includesthe gene for the cI⁴³⁴ repressor protein and its associated promoter andoperator sequence. cI⁴³⁴ represses a cI⁴³⁴⁻ lysogen; loss of the plasmidwill result in cell lysis. The distance between the 3' end of the P_(L)O_(L) promoter and operator sequence and the 5' end of the N utilizationsite is less than about 80 base pairs and the distance between the 3'end of the N utilization site and the 5' end of the ribosomal bindingsite is less than about 300 base pairs.

Another component of the vector is a first restriction enzyme sitepermitting replacement of the DNA sequence containing the ribosomalbinding site which follows thereafter. Numerous such sites may be used.Suitable sites include EcoRI.

Yet another component of the vector is a second restriction enzyme sitefor insertion of desired genes into the vector in phase with the ATGinitiation codon. Numerous such sites may be used. Suitable sitesinclude NdeI, ClaI, HindIII, SmaI, BglII, XbaI, SacI and AluI.

Generally it is desirable that the second restriction enzyme site alsofunction as the second restriction site necessary to permit replacementof the DNA sequence containing the ribosomal binding site. If the secondrestriction site is not also used for this purpose then the vector ofthis invention must also include a third restriction enzyme site afterthe ribosomal binding site but prior to the second restriction site.

A further component of the vector is a T₁ T₂ transcription terminationsequence. Preferably, the T₁ T₂ rRNA termination sequence is less thanabout 100 base pairs from the 3' end of the second restriction enzymesite. More preferably, the T₁ T₂ rRNA sequence is less than about 20base pairs from the 3' end of the second restriction enzyme site.

Yet a further component of the vector is that it has an origin ofreplication from a constitutive high copy number plasmid. Preferably,this origin of replication is ColEl. More preferably, the origin isplasmid pOP1Δ6 which has a restriction map shown in FIG. 7.

The vector also includes either the cI⁴³⁴ repressor gene which repressesa λimm434cI⁻ lysogen or a gene associated with a selectable oridentifiable phenotypic trait which is manifested when the vector ispresent in the host cell or both. When the cI⁴³⁴ repressor gene iscontained within a the host is prevented from λimm434cI⁻ propageinduction. Thus, there is no need to use expensive antibiotic selectionsalines when cI⁴³⁴ is present.

Preferably, when the cI⁴³⁴ gene is included on the vector, it is locatedafter the 3' end of the T₁ T₂ rRNA sequence.

Preferably, the vector contains two unique restriction enzyme sites. Thefirst site permits replacement of the DNA sequence containing theribosomal binding site. The second site permits insertion of the desiredgene into the vector in phase with the ATG initiation codon. In apresently preferred embodiment, EcoRI is the first restriction enzymesite and NdeI is the second restriction enzyme site.

The preferred host for use with the vector is Escherichia coli. Thepresently preferred strains are A1637, A1645 (c600 r⁻⁺ gal⁺ thr⁻ leu⁻lac⁻ Z⁻ (λcI857 ΔH1 ΔBam N+)), A2602, A2097, A1563 and A1645 (λi434cI⁻mini Tn10). A1645 (λi434cI⁻ mini Tn10) is presently the most preferredstrain for expression of animal growth hormone genes. It has beendeposited with the American Type Culture Collection in Rockville, Md.,U.S.A. containing plasmids as described more fully hereinafter. All ofthe deposits were made pursuant to the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms except thatpBR322 and pBRM are fully available from the American Type CultureCollection as ATCC Nos. 37017 and 37283, respectively, and D4 wasdeposited under ATCC No. 31826 in connection with the filing of a U.S.patent application.

A1645 (C600 r⁻ m⁺ gal⁺ l thr⁻ leu⁻ lac⁻ Z⁻ cI⁸⁵⁷ ΔH₁ ΔBam N+)) wasobtained from A1637 by selection for Gal⁺ (ability to ferment galactose)as well as loss of tetracycline resistance. It still contains the lambdaexpression system but part of the transposon has been removed byselection.

A2602 and A1563 are derived from SA500. Their phenotypes are SA500 his⁻ile⁻ gal⁺ 8(λCI857 ΔH1 ΔBam N+) and SA500 his⁻ ile⁻ gal⁺ Δ8 lac ZxA21(CI859 int2 xis1 nutL3 ΔH1), respectively. A2097 is derived from A1645.Its phenotype is A1645 lac ΔχA21 proC:Tn10.

A1645 (λi434cI.sup.⁻ was derived by infecting Escherichia coli strainA1645 containing a plasmid with λimm434cI³⁰⁰⁸ mini Tn Δ16 Δ17 at 30° C.Tetracycline resistant colonies were isolated and purified. The straincontaining plasmid pHG50 (FIG. 6) has been deposited with the AmericanType Culture Collection under ATCC No. 39805.

Preferably, the vector is a covalently closed circular double-strandedmolecule. However, it is not essential that the vector be covalentlyclosed.

The vector achieves its enhanced expression levels after the host cellis heated to a temperature at which the C_(I) repressor protein isdestroyed. A temperature above about 38° C. is effective for thispurpose and since it is desired that unnecessary heat damage to the hostcells be avoided to as great an extent as possible, it is generallydesirable that the temperature not exceed 42° C. by more than a fewdegrees.

One important component of the vector is the ribosomal binding site.Suitable sites are C_(II) from lambda bacteriophage having the sequence:##STR9## a mutant of C_(II) from lambda bacteriophage having thesequence: ##STR10## the major head protein gene of bacteriophage lambdahaving the sequence: ##STR11## the natural β-lactamase ribosomal bindingsite derived from pBR322;

a synthetic oligonucleotide having the sequence: ##STR12##

a synthetic oligonucleotide having the sequence: ##STR13## a naturalribosomal binding site derived from Bacillus thurengensis.

A DNA sequence which contains a gene associated with a selectable oridentifiable phenotypic trait which is manifested when the vector ispresent in the host cell is also a component of the vector. Suitablegenes include those associated with temperature sensitivity or drugresistance, e.g., resistance to ampicillin, chloroamphenicol ortetracycline.

Relative to vectors described previously, the vectors of this inventionmay be used to obtain enhanced expression of a wide variety of genesencoding desirable polypeptide products. Suitable genes include thoseencoding growth hormones, e.g., bovine, porcine, chicken or human growthhormones; superoxide dismutase; apoliprotein E or analogs of any of thepreceding. By analog is meant a polypeptide having the same activity asthe naturally occurring polypeptide but having one or more differentamino acids added or deleted, or both, at the N-terminus of thepolypeptide.

The vector may be formed by methods well known to those of ordinaryskill in the art to which the invention relates. Such methods aredescribed in greater detail in various publications identified herein,the contents of which are hereby incorporated by reference into thepresent disclosure in order to provide complete information concerningthe state of the art.

One presently preferred vector is derived by removing the met--phe bGHgene from plasmid p830-10A. The plasmid has the restriction map shown inFIG. 7 and has been deposited in strain A2097 with the American TypeCulture Collection under ATCC No. 39785.

Another presently preferred vector is derived by removing the rec bGHgene from plasmid pSAL-170/10. The plasmid has the restriction map shownin FIG. 8.

A third presently preferred vector is derived by removing the rec bGHgene from plasmid pSAL-210/4. The plasmid has the restriction map shownin FIG. 9.

The vectors of this invention, upon introduction into a host, may alsobe engineered to yield plasmids which produce analogs of bovine growthhormone. p830-10A (ATCC No. 39785), one example of such a plasmid, wasconstructed according to the scheme shown in FIG. 7. The analog itproduces has a methionine residue added to the aminoterminus of thephenylalanine form of natural bGH.

Other plasmids produce analogs which have the amino acid sequencemet--asp--gln added to N-terminus of the phenylalanine form of naturalbGH. These .plasmids include pSAL-130/5 (FIG. 8), pSAL-170/10 (FIG. 8)and pSAL-210/4 (FIG. 9).

Using the same approach other plasmids may be prepared by inserting intothe second restriction enzyme site of a vector according to theinvention a gene encoding a desired polypeptide.

Various host vector systems involve Escherichia coli strains A1637,A1645, A2606, A2097 or A1563 if the plasmid does not contain the cI⁴³⁴fragment and strain A1645 (λi434c⁻ mini Tn10) if the plasmid containsthe cI⁴³⁴ fragment. The host vector systems and plasmid described hereinmay be used to produce different polypeptides such as bovine, porcine,chicken and human growth hormones, human superoxide dismutase and humanapoliprotein E. To do so, the host vector system is grown under suitableconditions permitting production of polypeptide which is then recovered.

Suitable conditions involve growth of the host vector system for anappropriate period of time at about 42° C. Desirably, the period ofgrowth at 42° C. is about 1 to 5 hours. Suitable media include caseinhydrolysate.

Veterinary compositions may be prepared which contain effective amountsof bGH analog and a suitable carrier. Such carriers are well known tothose of ordinary skill in the art. The analogs may be administereddirectly or in the form of a composition to a cow in order to increasemilk or meat production.

EXAMPLES

The examples which follow are set forth to aid in understanding theinvention but are not intended to, and should not be construed to, limitits scope in any way. The examples do not include detailed descriptionsfor conventional methods employed in the construction of vectors, theinsertion of genes encoding polypeptides of interest into such vectorsor the introduction of the resulting plasmids into bacterial hosts. Suchmethods are well known to those of ordinary skill in the art and aredescribed in numerous publications including by way of example thefollowing:

J. Miller, Experiments in Molecular Genetics, Cold Spring HarborLaboratory, New York (1972).

T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning; ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1982).

Methods in Enzymology, vol. 65, "Nucleic Acids (Part 1)," edited byLawrence Grossman and Kivie Moldave, Academic Press, New York (1980).

Methods in Enzymology, vol. 68, "Recombinant DNA," edited by Ray Wu,Academic Press, New York (1981).

Methods in Enzymology, vol. 100, "Recombinant DNA (Part B)," edited byRay Wu, Lawrence Grossman and Kivie Moldave, Academic Press, New York(1983).

Methods in Enzymology, vol. 101, "Recombinant DNA (Part C)," edited byRay Wu, Lawrence Grossman and Kivie Mold ave, Academic Press, New York(1983).

Principles of Gene Manipulation, An Introduction to Genetic Engineering,2nd Edition, edited by R. W. Old and

S. B. Primrose, University of California Press (1981).

H. V. Bernard, et al., Gene (1979) 5, 59.

A. B. Oppenheim, et al., J. Mol. Biol. (1982) 158, 327.

E. Remaut, et al., Gene (1981) 15, 81.

EXAMPLE 1 p830-10A

The construction of p830-10A (ATCC No. 39785) is shown in FIG. 7 and isdescribed in the Description of the Figures. The plasmid p830-10A wasderived from the constitutive high copy number plasmid pOP1Δ6 (Gelfand,D. H., et al., PNAS (1978) 75, 5869; Meusing, et al., Cell (1981) 24,235-242). p7200-22, also shown in FIG. 7, was digested with ClaI and theClaI-ClaI fragment, which contains the λP_(L) promoter, the bGH gene andthe T₁ T₂ sequences was isolated. The ClaI-ClaI fragment was insertedinto the unique ClaI site of pOP1Δ6. (The plasmid p7200-22 is aderivative of pSAL 5600-1 (FIG. 10) in which a synthetic ClaI linker wasintroduced at the BglII site "upstream" of theλP_(L) promoter.)

Plasmid p830-10A was found to maintain the constitutive high copy numberphenotype even after induction of the λP_(L) promoter at about 42° C.This may be due to the presence of the T₁ T₂ termination sequences atthe 3' end of the bGH sequence which prevents formation of long mRNAtranscripts from the λP_(L) promoter which might interfere with othermRNA transcripts at the origin of replication of pOP1Δ6.

p830-10A was introduced into Escherichia coli strain A2097 bytransformation using methods known to those of ordinary skill in theart. This strain produces upon growth and induction an analog of bovinegrowth hormone having a methionine residue added to the amino terminusof the phenylalanine form of natural bGH. The amount of bGH analogproduced was about 37-43% of the total protein produced by the bacteriaas calculated by scanning Coomasie-stained SDS polyacrylamide gels. Themethods used to grow the strain, recover the bGH analog produced andpurify the bGH analog, are the same as those described for pSAL-170/10in Example 5.

                  TABLE I.sup.1                                                   ______________________________________                                        Plasmid       % bGH.sup.2                                                                              Remarks                                              ______________________________________                                        pRec 2/3      23         Amp.sup.R                                            pRO11         28         Amp.sup.R                                            pRO12         30-36      Amp.sup.R                                            pHG44         37-42      Amp.sup.R,T.sub.1 T.sub.2                            pHG50         37-42      Amp.sup.R,T.sub.1 T.sub.2 ;cI.sup.434                pSAL-130/5    39-44      Amp.sup.R ;CHCN;T.sub.1 T.sub.2                      pSAL-170/10   40-46      Tet.sup.R ;CHCN;T.sub.1 T.sub.2                      ______________________________________                                         .sup.1 The table summarizes the bGH expression levels of various plasmids     derived from pRO211. The plasmids pRec 2/3 and pRO11 are described in         copending, coassigned U.S. Pat. application Ser. No. 514,188, filed July      15, 1983.                                                                     .sup.2 Amount of bGH produced as percentage of total bacterial protein.       ABBREVIATIONS                                                                 CHCN = Constitutive high copy number                                          Amp.sup.R = Ampicillin resistance                                             Tet.sup.R = Tetracycline resistance                                           T.sub.1 T.sub.2 = Transcription termination sequences                         cI.sup.434 = Plasmid stabilization cI.sup.434 system                     

EXAMPLE 2

A general utility expression vector may be derived from the plasmidp8300-10A (FIG. 7, ATCC No. 39785) by excision of the bGH gene. A vectorso derived has numerous advantages over previously described expressionvectors including:

1. Extremely High Levels of Expression

The vector is capable of directing expression of foreign proteins inEscherichia coli at levels as high as 44% of the total cellular protein.constitutive high copy number

2. Constitutive High Copy Number

The vector maintains a constitutively high copy number of about 200-300copies per cell. This is in distinction to other λP_(L) expressionvectors which are present in lower copy numbers. The high copy numbercontributes to higher levels of expression.

3. Transcription Termination Signals

The vector which may be obtained by excision of bGH sequence fromp8300-10A contains the T₁ T₂ transcription termination signals placed"downstream" from the λP_(L) promoter and C_(II) ribosomal binding site.The high levels of expression are due in part to the presence of the T₁T₂ transcription terminators at the end of the inserted genetranscription of N-modified RNA polymerase. Thus the transcriptionterminators prevent the λP_(L) -controlled transcription of undesiredplasmid proteins, thereby enhancing the relative yields of the desiredprotein. Furthermore the presence of the T₁ T₂ transcription terminationsignals prevents long mRNA transcripts through the plasmid origin ofreplication. This enhances the stability of the high copy phenotype.

Similar high copy number plasmids containing the λP_(L) promoter butlacking the transcription termination sequences are unstable and tend tolose the high copy number phenotype.

4. Replaceable Ribosomal Binding Site

p8300-10A contains a unique EcoRI site which is located "upstream" ofthe ribosomal binding site, and an NdeI site located "downstream" of theribosomal binding site. Thus, the ribosomal binding site is bounded bytwo unique restriction sites. This enables facile excision of thepresent ribosomal binding site (the λC_(II) ribosomal binding site) andsubstitution of virtually any other natural or synthetic ribosomalbinding site without altering other features of the plasmid. Thisgreatly facilitates optimal expression of desired polypeptides.

5. Thermoinducible Regulation of Expression

The λP_(L) promoter is inactive when the C_(I) repressor is bound to it.The cI857 repressor is thermosensitive, that is, it binds to thepromoter at 30° C. but is inactivated at 42° C. Thus, by increasing thetemperature of fermentation to 42° C. the host bacteria are induced toproduce the desired protein.

The advantages of such a system include the following:

(a) A foreign protein which is toxic to Escherichia coli can be producedlate in the fermentation

process thus avoiding early cell death,

(b) Overproduction of a protein may stabilize the protein and preventproteolytic degradation. (Cheng, Y. E., et al., Gene (1981) 14, 121).

Thus, "instantaneous" overproduction using a tightly regulated promotersuch as λP_(L) may be preferable to continuous low level production.

6. Simplified Induction Protocol

Protein production by the plasmids described in this patent applicationand in copending, coassigned U.S. patent application Ser. No. 514,188 isregulated by the thermosensitive cI⁸⁵⁷ repressor.

The induction protocol required by the plasmids described in thecopending, coassigned application involved induction at 42° C. followedby growth at 38° C. In contrast, the optimal induction of proteinsynthesis when using the plasmid p8300-10A or pSAL-130/15 or theirplasmid derivatives involved induction at 42° C. followed by growth atthe same temperature, i.e., 42° C. This eliminates the need to cool thefermentor.

7. Ribosome Binding Site and Initiation Codon

This expression vector contains a strong procaryotic ribosomal bindingsite (RBS) as well as a translation initiation codon (ATG). Thus, anyeucaryotic gene may be cloned without adding the initiation codon.Furthermore, the efficient RBS increases levels of expression. Theribosome binding site is the λ C_(II) ribosomal binding site. Thesequence of the ribosomal binding site is: ##STR14## One base pair isdifferent from the ribosomal binding site found in the wild type λ.

8. Convenient Restriction Site

The expression vector has a unique NdeI restriction site which containswithin the site the ATG initiation codon. This permits properpositioning of the desired gene. The unique NdeI site is foundimmediately after the ribosomal binding site.

9. Nut Site

N protein, which is provided by the host, binds the Nut site on theexpression vector and thereby prevents termination of transcription atthe tRI site or premature transcription termination within the clonedgene.

Strains

Suitable hosts for the described vectors and plasmids are strains ofEscherichia coli suitable for transformation, including A1637, A2602,A1563, A1645 (c600 r⁻ m⁺ gal⁺ thr⁻ leu⁻ lac⁻ b1 (λcI⁸⁵⁷ ΔH1 ΔBamHI N⁺))and A2097 (A1645 lac ΔχA21 proC::Tn 10).

EXAMPLE 3 pSAL-130/5

The construction of pSAL-130/5 is shown in FIG. 8 and described in theDescription of the Figures. pSAL-130/5 was obtained from p8300-10A (ATCCNo. 39785) by replacing the met--phe bGH gene with the met--asp--gln bGHgene. The met--asp--gln bGH gene was obtained from plasmid pHG44 (FIG.6) (ATCC No. 39806) by NdeI and HindIII digestion.

pSAL-130/5 was introduced into Escherichia coli strain A1645 bytransformation using methods known to those of ordinary skill in theart. This strain produces upon growth and induction an analog of bovinegrowth hormone (bGH) having the amino acid sequence met--asp--gln addedto the N-terminus of the phenylalanine form of natural bGH. The amountof bGH analog produced by pSAL-130/5 was about 39-44% of the totalprotein produced by the bacteria as calculated by scanning Coomasieblue-stained SDS polyacrylamide gels (Table I). The methods used to growthe strain, recover the bGH analog produced and purify the bGH analogare the same as those described for pSAL-170/10 in Example 5.

EXAMPLE 4 pSAL-170/10

The construction of pSAL 170/10 is shown in FIG. 8 and described in theDescription of the Figures.

pSAL 170/10 was introduced into Escherichia coli strain A1645 bytransformation using known methods. This strain produces upon growth andinduction an analog of bGH having the amino acid sequence met--asp--glnadded to the amino terminus of the phenylalanine form of natural bGH.The amount of the bGH analog produced by pSAL-170/10 was about 40-46% ofthe total protein produced by the bacteria as calculated by scanning theCoomasie-stained SDS polyacrylamide gels. (Table I).

EXAMPLE 5 Growth of pSAL-170/10 I. Stock Cultures

Stock cultures of pSAL-170/10 were grown on casein medium (seeInoculum), then diluted two-fold with freezing medium and stored at -80°C. Freezing medium contains per 500 ml:

    ______________________________________                                        K.sub.2 HPO.sub.4     6.3    gr                                               KH.sub.2 PO.sub.4     1.8    gr.                                              Na Citrate            0.45   gr                                               MgSO.sub.4.7H.sub.2 O 0.09   gr                                               (NH.sub.4).sub.2 SO.sub.4                                                                           0.9    gr                                               Glycerol              44.0   gr                                               ______________________________________                                    

II. Inoculum

The inoculum was propagated in 20 g/l casein hydrolysate, 10 g/l yeastextract and 2 g/l NaCl. Sterile medium in a shake flask was inoculatedfrom stock culture and incubated 15 hours on a shaker at 30° C. andapproximately 200 r.p.m. As needed subsequent stages in inoculumpropagation were carried our in stirred aerated fermenters. Sterilemedium was inoculated with 2-10% inoculum and incubated 15 hours at 30°C., pH 7±0.5 with agitation and aeration to maintain a dissolved oxygenlevel above 20% air saturation.

III Production

7 The production medium contains:

    ______________________________________                                        Casein hydrolysate     20    g/l                                              Yeast extract          10    g/l                                              K.sub.2 HPO.sub.4      2.5   g/l                                              MgSO.sub.4.7H.sub.2 O  1     g/l                                              NaCl                   5     g/l                                              Biotin                 0.1   mg/l                                             Thiamine               1     mg/l                                             Trace elements solution                                                                              3     ml/l                                             ______________________________________                                    

The medium also contains 12.5 mg/liter tetracycline. The tetracycline isoptional for production but is always found in the medium used forgrowing the inoculum.

Biotin, thiamine and antibiotics in concentrated solution were filtersterilized separately and added to the sterile production medium beforeinoculation. Sterile glucose solution was added initially to supply 10g/1. At the induction step another 10 g/l of glucose was added.

The trace elements solution contains

    ______________________________________                                        FeCl.sub.3           16     g/l                                               ZnCl.sub.2.4H.sub.2 O                                                                              2      g/l                                               CoCl.sub.2.6H.sub.2 O                                                                              2      g/l                                               Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                      2      g/l                                               CaCl.sub.2.2H.sub.2 O                                                                              1      g/l                                               CuCl.sub.2           1      g/l                                               H.sub.3 BO.sub.3     0.5    g/l                                               Conc. HCl            100    ml/l                                              ______________________________________                                    

The medium is inoculated with 0.5-10% inoculum culture and incubated at30° C. Agitation-aeration rates are set to maintain a dissolved oxygenlevel above 20% air saturation. The pH is maintained at 7±0.2 with NH₃.Once cell concentration reaches about 3.5 g/l (OD₆₆₀ =10) induction isstarted.

The temperature is raised to 42° C. and maintained at 42° C. for 1-5hours. The culture is then chilled and cells are recovered bycentrifugation for hormone purification.

Recovery of bGH

Thirteen kilograms of bacterial cells (wet cake) are resuspended in 5volumes of a solution containing 50 mM sodium phosphate buffer (pH 7.4),50 mM EDTA and 100 mM NaCl, using a Polytron (Kinematica) blender, whilecontrolling the blender's speed to minimize foaming. The homogenoussuspension is continuously passed through a Dynomill cell disruptor KD5(Willy A. Bachofen, Basel) at a rate of 80 liter per hour and thehomogeneous suspension of disrupted cells clarified by centrifugation ina CEPA 101 centrifuge at a flow rate of 45 liter per hour. Theprecipitate from the centrifugation step is collected and resuspended in15.5 liters of 50 mM sodium phosphate buffer (pH 7.4) containing 50 mMEDTA. Lysozyme is added to a final concentration of 0.05 mg/ml and thesuspension incubated for 16 hours at 37° C. Triton X-100 is added to afinal concentration of 1%. The suspension is then incubated for 30minutes at room temperature, sonicated in a continuous flow cellsonificator (Heat System) at a rate of 18 liters per hour andcentrifuged in a CEPA 101 centrifuge. The precipitate is collected,resuspended in 50 mM sodium phosphate buffer (pH 7.4), sonicated asabove, and centrifuged in a CEPA 101 centrifuge. The cells areresuspended in 15.5 liters of 50 mM sodium phosphate buffer (pH 7.4)containing 50 mM EDTA and 100 mM NaCl and twice precipitated andresuspended in 15.5 liters of distilled water. The precipitate iscollected by centrifugation and stored at -20° C.

Purification Of bGH

The precipitate is resuspended in 30-40 liters distilled water andsolubilized by titration with 0.5N NaOH to pH 11.8. The solution is thencontinuously sonicated and clarified by centrifugation in CEPA 101centrifuge if necessary, or filtered through Whatman No. 1 paper.

The clarified protein solution (32.6 liters containing 297,000 OD's at280 nm) is divided into separate portions (6×5.4 liters) each containing50,000-60,000 OD's. Each portion is ultrafiltered separately through aMillipore Pellicon ultrafilter equipped with three 100,000 molecularweight cutoff cassettes (type PTHK) of 5 ft² area each. A 5.4 literportion is concentrated to 1 liter retentate volume. The ultrafiltrateis collected and saved. The retentate is diluted back to its originalvolume with fresh 10 mM Borate buffer, pH 11.8, and mixed well. Thebatch is concentrated again to 1 liter retentate volume. Theultrafiltrate is collected and combined with the first ultrafiltrate.When the running total of the OD's in the ultrafiltrates equals 20% ofthe OD's initially charged to the ultrafilter, the retentate volume onthe next concentration step is taken to 0.5 liters instead of 1 liter.The cycle of concentration and dilution with 10 mM Borate buffer iscontinued until the ultrafiltrate from a retentate volume of 0.5 litershas an absorbance at 280 nm (1-cm cell) of less than 0.1. This normallytakes between 9 and 12 cycles of concentration and dilution. The finalretentate is discarded.

All ultrafiltrates are combined and adjusted to pH 9.0 with 6N HCl. Theother 5.4-liter portions are ultrafiltered in the same fashion, and allpH adjusted ultrafiltrates are combined. A typical run produces a totalof 380 liters of ultrafiltrates with an absorbance of 0.26 equivalent to100,000 OD's and requires 24 to 40 hours to complete.

The combined ultrafiltrates (380 liters containing 100,000 OD's at 280nm) from the 100K ultrafiltration step are loaded onto a Sepharose CL-6BDEAE ion-exchange column at a linear flow velocity of 23 cm/hr (25liter/hr). The 37-cm diameter 15-cm high column is washed with two bedvolumes (32 L) of 10 mM Borate buffer at pH 9.0. The eluate from theloading and washing steps is discarded. A step change in eluent to 10 mMBorate, 100 mM sodium chloride, pH 9, displaces the bGH off the column.The elution flow velocity is 23 cm/hr. The progress of the run ismonitored by following absorbance of the eluate at 280 nm. The bGH peakis collected in 4 to 5 bed volumes (84 liters containing 43,000 OD's at280 nm) and then concentrated to approximately 10 mg/ml using aMillipore Pellicon ultra-filtration device with a 10,000 molecularweight cutoff cassettes. The solution is then lyophilized. The yield isapproximately 70 g of pure bGH.

EXAMPLE 6 Activity Of bGH Analog Produced By pSAL-170/10 1.Radioimmunoassay Comparison of bGH Analog with Natural bGH

A solution containing 100 ng/ml bGH analog was prepared in phosphatebuffered saline (1% BSA). This solution was diluted serially toconcentrations of 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 ng/1.Duplicate 0.1 ml aliquots of these solutions were submitted to RIA usinga double antibody procedure. The dilution curve was comparable to thatobtained with natural bGH.

2. Radioreceptor Binding Assay

A radioreceptor binding assay was performed with rabbit liver membranesas described by Tushima, T. and Freisen, H. G., (Y. Chin., Endocr.Metab. (1973), 37; 3) using ¹²⁵ I-bGH as the tracer and authentic bGHsolutions for the construction of calibration curves. Samples wereincubated in triplicate for two hours at room temperature in 0.3 ml ofassay buffer (50 mM Tris, 15 mM CaCl₂ and 5 mg/ml bovine serum albumin,pH 7.6). The tubes contained ¹²⁵ I-bGH (20,000 cpm of preparation of30-60 μci/μg), 150-250 μg liver membrane protein and either natural bGH(1-100 ng) or extracts of bacterial bGH. The result demonstrated thatthe bGH activity of the bGH analog is comparable to that of natural bGH.

3. Tibia Test

The bioactivity of the pRO12 produced bGH analog recovered frombacterial cells according to Example 5 was evaluated by a tibia test.(Parlow, A. F., et al., Endocrinology (1965) 77:1126). Rats werehypophysectomized at 28-30 days of age, then kept for 10-14 days withouttreatment. Bovine growth hormone derived from bovine pituitaries or fromrecombinant Escherichia coli was dissolved in 0.15M NaCl+0.01M borate,pH 10.0. Rats (4-7 per group) received daily subcutaneous injections ofbGH solutions (5-125 μg/day in 0.2 cc) for 5 days while kept on a normaldiet (Purina Rat-Chow and water adlibitum). The animals were sacrificedon the 6th day, their foreleg knee-bones taken out, cut longitudinally,fixed with acetone and stained with 2% AgNO₃. The width of theepiphyseal plates was measured by observation through a dissectingbinocular (Nikon). Mean values (40 readings per rat) were used for theconstruction of long dose-response curves. The results demonstrated thatthe bGH activity of the pSAL-170/10-produced bGH analog is comparable tothat of natural bGH.

What is claimed is:
 1. A plasmid for production of a bovine growthhormone polypeptide analog having the amino acid methionine attached tothe N-terminus of the phenylalanine form of the naturally-occurringpolypeptide, said analog having substantially the same biologicalactivity as the naturally-occurring polypeptide, designated p8300-10Ahaving the restriction map shown in FIG. 7 and deposited under ATCCAccession No.
 39785. 2. A plasmid for production of a bovine growthhormone polypeptide analog having the amino acids methionine, asparticacid and glutamine attached to the N-terminus of the naturally-occurringpolypeptide, said analog having substantially the same biologicalactivity as the naturally-occurring polypeptide, designated pSAL-130/5having the restriction map shown in FIG.
 8. 3. A plasmid for productionof a bovine growth hormone polypeptide analog having the amino acidsmethionine, aspartic acid and glutamine attached to the N-terminus ofthe naturally-occurring polypeptide, said analog having substantiallythe same biological activity as the naturally-occurring polypeptide,designated pSAL 170/10 having the restriction map shown in FIG.
 8. 4. Ahost plasmid system for production of met--phe bovine growth hormonehaving the amino acid methionine attached to the N-terminus of thephenylalanine form of the naturally-occurring polypeptide whichcomprises the plasmid of claim 1 in a suitable E. coli host.
 5. A hostplasmid system of claim 4, wherein the Escherichia coli host strain isselected from the group consisting of strain A1637, A1645, A2602, A2097or A1563.
 6. A method for producing bovine growth hormone polypeptideanalog which comprises growing the hose plasmid system of claim 4 undersuitable conditions permitting production of the bovine growth hormonepolypeptide analog and recovering the resulting bovine growth hormoneanalog.
 7. A hose plasmid system for production of bovine growth hormoneanalog having the amino acids methionine, aspartic acid and glutamineattached to the N-terminus of the naturally-occurring polypeptide whichcomprises the plasmid of claims 2 or 3 in a suitable E coli host.
 8. Ahost plasmid system of claim 7, wherein the Escherichia coli host strainis selected from the group consisting of strain A1637, A1645, A2602,A2097 or A1563.
 9. A method for producing bovine growth hormonepolypeptide analog which comprises growing the hose plasmid system ofclaim 7 under suitable conditions permitting production of the bovinegrowth hormone polypeptide analog and recovering the resulting bovinegrowth hormone analog.